Disulfide-Containing Alkyne Linking Agents

ABSTRACT

Described are improved disulfide-containing alkyne linking agents having have branched disulfides. The improved linking agents exhibit improved stability. The linking agents are useful for attachment of oligonucleotides to targeting groups or delivery agents.

BACKGROUND

Synthetic oligonucleotides, such as antisense molecules, aptamers,ribozymes and RNA interference (RNAi) molecules, are increasingly usedin biomedical research, diagnostics and therapeutics. These syntheticoligonucleotides have been used to inhibit or knock-down expression of agene in vitro, in situ, and in vivo in a sequence dependent manner.

It is frequently useful to attach or link targeting ligands or otherpharmacological modifiers to synthetic oligonucleotides, especially fortherapeutic in vivo delivery. To be useful, the linkage chemistry shouldbe modular, so that it is readily adaptable to different syntheticoligonucleotides as well as different targeting ligands andpharmacological modifiers. In addition, the linkage chemistry shouldhave simple reaction conditions, be efficient (i.e. give high chemicalyields), not require toxic or other detrimental products, and notproduce toxic or other detrimental byproducts. The linkage chemistryshould also be stable outside of the target cell, such as incirculation, subcutaneous space, or extracellular space, but be readilycleavable at the final site of action, such as inside the target cell.

An example of such a reaction useful in linking syntheticoligonucleotides to targeting ligands or other pharmacological modifiersis the cycloaddition of cyclic alkynes and azides, which is one of thereactions known as “click reactions” or “click chemistry”. In clickreactions, two separate molecular entities, one charged with an azideand one charged with a strained cycloalkyne, will spontaneously combineinto a single molecule by a reaction called strain-promoted azide-alkynecycloaddition (SPAAC). The reaction is mild in nature, rapid, andhigh-yielding and occurs at about physiological pH, in water, and in thevicinity of biomolecular functionalities. This reaction has become aversatile tool for bioorthogonal labeling and imaging of biomolecules(e.g. proteins, lipids, glycans and the like), proteomics and materialsscience. The cycloaddition reaction proceeds spontaneously, in theabsence of a catalyst. Metal-free cycloadditions are also referred to as“metal-free click reactions”. The power of SPAAC for bioorthogonallabeling lies in the fact that an isolated cyclic alkyne or azide isfully inert to biological functionalities, such as for example amines,thiols, acids or carbonyls, but in combination undergoes rapid andirreversible cycloaddition, leading to a stable triazole conjugate. Forexample, azido-modified proteins, obtained by expression in auxotrophicbacteria, genetic engineering or chemical conversion, can be cleanlylabeled with biotin, fluorophores, PEG-chains or other functionalitiesupon simply stirring the azido-protein with a cyclooctyne conjugate.Moreover, the small size of azide has proven highly useful forapplication of SPAAC in the imaging of specific biomolecules by means ofthe chemical reporter strategy.

We have found the alkyne disulfides currently available, such asDibenzocyclooctyne (DBCO)—S—S—NHS,

offer insufficient stability during modification of compounds leading topoor synthetic yields and the presence of undesired impurities. We nowdescribe improved alkyne disulfides having improved stability propertiesresulting in improved yields and purity.

SUMMARY

Described herein are cyclooctyne-alkyl disulfide compounds having thestructure represented by:

wherein R¹ and/or R² are alkyl groups, L¹ and L² are linkers, X is acyclooctyne, and Y comprises a reactive group, syntheticoligonucleotide, or RNAi agent. In some embodiments, R¹ is an alkylgroup and R² is a hydrogen. In some embodiments, R² is an alkyl groupand R¹ is a hydrogen. In some embodiments, the alkyl group is a methylgroup or ethyl group. In some embodiments, Y comprises an RNAi agent.

Described herein are cyclooctyne-alkyl disulfide compounds having thestructure represented by:

wherein R¹, R^(1′), R² and/or R^(2′) are alkyl groups, X comprises acyclooctyne, and Y comprises a reactive group or a syntheticoligonucleotide. In some embodiments, R¹ and R^(1′) are alkyl groups andR² and R^(2′) are hydrogens. In some embodiments, R² and R^(2′) arealkyl groups and R¹ and R^(1′) are hydrogens. In some embodiments, R¹,R^(1′), R², and R^(2′) are alkyl groups. In some embodiments, R¹ is analkyl groups and R^(1′), R², and R^(2′) are hydrogens. In someembodiments, R^(1′) are alkyl groups and R¹, R², and R^(2′) arehydrogens. In some embodiments, R² are alkyl groups and R¹, R^(1′), andR^(2′) are hydrogens. In some embodiments, R^(2′) are alkyl groups andR¹, R^(1′), and R² are hydrogens. In some embodiments, R¹, R^(1′), andR² are alkyl groups R^(2′) is a hydrogen. In some embodiments, R¹,R^(1′), and R^(2′) are alkyl groups R² is a hydrogen. In someembodiments, R¹, R², and R² are alkyl groups R^(1′) is a hydrogen. Insome embodiments, R^(1′), R², and R^(2′) are alkyl groups R¹ is ahydrogen. In some embodiments, the alkyl groups are methyl groups. Insome embodiments, Y comprises an RNAi agent.

Described herein are cyclooctyne-alkyl disulfide compounds having thestructure represented by:

wherein R³ is C(R⁵R⁶) wherein R⁵ is hydrogen, methyl or ethyl, and R⁶ ishydrogen, methyl or ethyl, R⁴ is C(R⁷R⁸) wherein R⁷ is hydrogen, methylor ethyl, and R⁸ is hydrogen, methyl or ethyl, at least one of R⁵, R⁶,R⁷, and R⁸ is methyl or ethyl, L¹ is a first linker, L² is a secondlinker, and Y comprises a reactive group, synthetic oligonucleotide orRNAi agent. In some embodiments, R⁵ or R⁶ is methyl or ethyl and R⁷ andR⁸ are both hydrogen. In some embodiments, R⁷ or R⁸ is methyl or ethyl,and R⁵ and R⁶ are both hydrogen. In some embodiments, R⁵ is CH₃, and R⁶,R⁷ and R⁸ are each hydrogen. In some embodiments, R⁷ is CH₃, and R⁵, R⁶and R⁸ are each hydrogen. In some embodiments, Y comprises an RNAiagent.

Described herein are cyclooctyne-alkyl disulfide compounds having thestructure represented by:

wherein R³ is C(R⁵R⁶) wherein R⁵ is hydrogen, methyl or ethyl, and R⁶ ishydrogen, methyl or ethyl, R⁴ is C(R⁷R⁸) wherein R⁷ is hydrogen, methylor ethyl, and R⁸ is hydrogen, methyl or ethyl, at least one of R⁵, R⁶,R⁷, and R⁸ is methyl or ethyl, L¹ is a linker, and Y comprises areactive group, synthetic oligonucleotide or RNAi agent. In someembodiments, R⁵ or R⁶ is methyl or ethyl, and R⁷ and R⁸ are bothhydrogen. In some embodiments, R⁷ or R⁸ is methyl or ethyl, and R⁵ andR⁶ are both hydrogen. In some embodiments, R⁵ is CH3, and R⁶, R⁷ and R⁸are each hydrogen. In some embodiments, R⁷ is CH₃, and R⁵, R⁶ and R⁸ areeach hydrogen. In some embodiments, Y comprises an RNAi agent.

Described herein are compounds having the structures represented by:

wherein R is a reactive group, NHS reactive group, syntheticoligonucleotide, or RNAi agent.

Described herein are compounds having the structure represented by:

wherein R comprises a reactive group, NHS reactive group, syntheticoligonucleotide, or RNAi agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Reaction scheme illustrating synthesis of DBCO-alkane-SS—NHS.

FIG. 2. Reaction scheme illustrating synthesis of DBCO-methyl-SMPT-NHS.

FIG. 3. Graphs showing ESI Mass Spec analysis of DBCO-methyl-SMPT-NHS.

FIG. 4. Graph showing H1 NMR of DBCO-methyl-SMPT-NHS.

FIG. 5. Disclosed embodiments of certain cyclooctyne-alkyl-S—Scompounds. R comprises a reactive group, NHS reaction group, syntheticoligonucleotide, or RNAi agent. n and m are integers from 0 to 10.

FIG. 6. Disclosed embodiments of certain cyclooctyne-alkyl-S—Scompounds. R comprises a reactive group, NHS reaction group, syntheticoligonucleotide, or RNAi agent.

FIG. 7. RP-HPLC chromatogram of the reaction mixture of the RNAi agentsense strand conjugate prepared with an unbranched DBCO disulfidemodifier (DBCO-disulfide-NHS ester).

FIG. 8. RP-HPLC chromatogram of the reaction mixture of the RNAi agentconjugate prepared with DBCO—NHS ester.

FIG. 9. RP-HPLC chromatogram of the reaction mixture of the RNAi agentsense strand conjugate prepared with DBCO-Alkane(methyl)-disulfidemodifier.

FIG. 10. RP-HPLC chromatogram of the reaction mixture of the RNAi agentsense strand conjugate prepared with BranchedDBCO-Alkane(dimethyl)-disulfide modifier.

FIG. 11. RP-HPLC chromatogram of the reaction mixture of the RNAi agentconjugate prepared with the DBCO-disulfide-methyl-SMPT.

FIG. 12. Disclosed embodiments of certaincyclooctyne-alkyl-S—S-oligonucleotide compounds R comprises anoligonucleotide or RNAi agent. The phosphate groups shown may be part ofa synthetic oligonucleotide or RNAi agent.

DETAILED DESCRIPTION

Novel compounds comprising cyclooctynes and branched disulfides, theirsynthesis, and methods of use thereof, are disclosed herein. Theimproved compounds disclosed herein exhibit improved stability withrespect to previously described compositions containing cyclooctynes.

Disclosed herein are cyclooctyne-alkyl-disulfide-modifiedoligonucleotides, their synthesis, and methods of use thereof. Thecyclooctyne-alkyl-disulfide-modified oligonucleotides are readilyattached to a compound of interest such as a targeting ligand, lipid,cholesterol, delivery agent (such as an endosomolytic polymer), orpharmacological modifier. The cyclooctyne-alkyl-disulfide-modifiedoligonucleotides are more readily synthesized with improved yield, andhave fewer impurities than corresponding cyclooctyne-disulfide-modifiedoligonucleotides.

In some embodiments, the cyclooctyne-alkyl disulfide compounds have thestructure represented by:

wherein

-   -   R¹ is an alkyl group and R² is hydrogen, or R¹ is hydrogen and        R² is an alkyl group, or R¹ and R² are alkyl groups.    -   X comprises a cyclooctyne,    -   Y comprises a reactive group or a synthetic oligonucleotide.

X and Y may be attached via linkers. In some embodiments, an alkyl groupis methyl or ethyl. In some embodiments, the alkyl group is methyl. Insome embodiments, Y comprises an RNAi agent.

In some embodiments, the cyclooctyne-alkyl disulfide compounds have thestructure represented by:

wherein

-   -   one or more of R¹, R^(1′), R2, and R2′ alkyl groups,    -   X comprises a cyclooctyne,    -   Y comprises a reactive group or a synthetic oligonucleotide.

X and Y may be attached via linkers. In some embodiments, the alkylgroups are independently methyl or ethyl. In some embodiments, the alkylgroup is methyl. In some embodiments, R¹ and R^(1′) are alkyl groups andR² and R^(2′) are hydrogens. In some embodiments, R² and R^(2′) arealkyl groups and R¹ and R^(1′) are hydrogens. In some embodiments, R¹,R^(1′), R², and R^(2′) are alkyl groups. In some embodiments, R¹ is analkyl groups and R^(1′), R², and R^(2′) are hydrogens. In someembodiments, R^(1′) are alkyl groups and R¹, R², and R^(2′) arehydrogens. In some embodiments, R² are alkyl groups and R¹, R^(1′), andR^(2′) are hydrogens. In some embodiments, R^(2′) are alkyl groups andR¹, R^(1′), and R² are hydrogens. In some embodiments, R¹, R^(1′), andR² are alkyl groups R^(2′) is a hydrogen. In some embodiments, R¹,R^(1′), and R^(2′) are alkyl groups R² is a hydrogen. In someembodiments, R¹, R², and R² are alkyl groups R^(1′) is a hydrogen. Insome embodiments, R^(1′), R², and R^(2′) are alkyl groups R¹ is ahydrogen. In some embodiments, Y comprises an RNAi agent.

In some embodiments, the cyclooctyne-alkyl disulfide compounds have thestructure represented by:

wherein

-   -   R¹ is an alkyl group and R² is hydrogen, or R¹ is hydrogen and        R² is an alkyl group, or R¹ and R² are alkyl groups,    -   L¹ and L² are linkers,    -   X comprises a cyclooctyne, and    -   Y comprises a reactive group or a synthetic oligonucleotide.

In some embodiments, the alkyl group is methyl or ethyl. In someembodiments, the alkyl group is methyl. In some embodiments, Y comprisesan RNAi agent.

In some embodiments, the cyclooctyne-alkyl disulfide compounds have thestructure represented by:

wherein

-   -   one or more of R¹, R^(1′) R² and R^(2′) are alkyl groups,    -   L¹ is a first linker,    -   L² is a second linker,    -   X comprises a cyclooctyne, and    -   Y comprises a reactive group or a synthetic oligonucleotide.

In some embodiments, the alkyl groups are independently methyl or ethyl.In some embodiments, the alkyl group is methyl. In some embodiments, R¹and R^(1′) are alkyl groups and R² and R^(2′) are hydrogens. In someembodiments, R² and R^(2′) are alkyl groups and R¹ and R^(1′) arehydrogens. In some embodiments, R¹, R^(1′), R², and R^(2′) are alkylgroups. In some embodiments, R¹ is an alkyl groups and R^(1′), R², andR^(2′) are hydrogens. In some embodiments, R^(1′) are alkyl groups andR¹, R², and R^(2′) are hydrogens. In some embodiments, R² are alkylgroups and R¹, R^(1′), and R^(2′) are hydrogens. In some embodiments,R^(2′) are alkyl groups and R¹, R^(1′), and R² are hydrogens. In someembodiments, R¹, R^(1′), and R² are alkyl groups R^(2′) is a hydrogen.In some embodiments, R¹, R^(1′), and R^(2′) are alkyl groups R² is ahydrogen. In some embodiments, R¹, R², and R² are alkyl groups R^(1′) isa hydrogen. In some embodiments, R^(1′), R², and R^(2′) are alkyl groupsR¹ is a hydrogen. In some embodiments, Y comprises an RNAi agent.

In some embodiments, the cyclooctyne-alkyl disulfide compounds have thestructure represented by:

wherein

-   -   R³ is C(R⁵R⁶) wherein R⁵ is hydrogen, methyl or ethyl, and R⁶ is        hydrogen, methyl or ethyl,    -   R⁴ is C(R⁷R⁸) wherein R⁷ is hydrogen, methyl or ethyl, and R⁸ is        hydrogen, methyl or ethyl,    -   at least one of R⁵, R⁶, R⁷, and R⁸ is methyl or ethyl,    -   L¹ is a first linker,    -   L² is a second linker, and    -   Y comprises a reactive group or a synthetic oligonucleotide.

In some embodiments, R⁵ or R⁶ is methyl or ethyl, and R⁷ and R⁸ are bothhydrogen. In some embodiments, R⁵ is methyl or ethyl, and R⁶, R⁷ and R⁸are hydrogen. In some embodiments, R⁶ is methyl or ethyl, and R⁵, R⁷ andR⁸ are hydrogen. In some embodiments, R⁵ and R⁶ are independently methylor ethyl, and R⁷ and R⁸ are both hydrogen. In some embodiments, R⁷ or R⁸is methyl or ethyl, and R⁵ and R⁶ are both hydrogen. In someembodiments, R⁷ is methyl or ethyl, and R⁸, R⁵ and R⁶ are hydrogen. Insome embodiments, R⁸ is methyl or ethyl, and R⁷, R⁵ and R⁶ are hydrogen.In some embodiments, R⁷ and R⁸ are independently methyl or ethyl, and R⁵and R⁶ are both hydrogen. In some embodiments, R⁵ is CH₃, and R⁶, R⁷ andR⁸ are each hydrogen. In some embodiments, R⁷ is CH₃, and R⁵, R⁶ and R⁸are each hydrogen. In some embodiments, the synthetic oligonucleotide isan RNAi agent.

In some embodiments, the cyclooctyne-alkyl disulfide compounds have thestructure represented by:

wherein

-   -   R³ is C(R⁵R⁶) wherein R⁵ is hydrogen, methyl or ethyl, and R⁶ is        hydrogen, methyl or ethyl,    -   R⁴ is C(R⁷R⁸) wherein R⁷ is hydrogen, methyl or ethyl, and R⁸ is        hydrogen, methyl or ethyl,    -   at least one of R⁵, R⁶, R⁷, and R⁸ is methyl or ethyl,    -   L¹ is a linker, and    -   Y comprises a reactive group or a synthetic oligonucleotide.

In some embodiments, R⁵ or R⁶ is methyl or ethyl, and R⁷ and R⁸ are bothhydrogen. In some embodiments, R⁵ is methyl or ethyl, and R⁶, R⁷ and R⁸are hydrogen. In some embodiments, R⁶ is methyl or ethyl, and R⁵, R⁷ andR⁸ are hydrogen. In some embodiments, R⁵ and R⁶ are independently methylor ethyl, and R⁷ and R⁸ are both hydrogen. In some embodiments, R⁷ or

R⁸ is methyl or ethyl, and R⁵ and R⁶ are both hydrogen. In someembodiments, R⁷ is methyl or ethyl, and R⁸, R⁵ and R⁶ are hydrogen. Insome embodiments, R⁸ is methyl or ethyl, and R⁷, R⁵ and R⁶ are hydrogen.In some embodiments, R⁷ and R⁸ are independently methyl or ethyl, and R⁵and R⁶ are both hydrogen. In some embodiments, R⁵ is CH₃, and R⁶, R⁷ andR⁸ are each hydrogen. In some embodiments, R⁷ is CH₃, and R⁵, R⁶ and R⁸are each hydrogen. In some embodiments, the synthetic oligonucleotide isan RNAi agent.

In some embodiments, the cyclooctyne-alkyl-disulfide-amine reactivegroup compounds have the structure represented by:

wherein R comprises a reactive group selected from the group comprising:activated ester, NHS, TFP, PFP, tetrazine, norbornenes,trans-cyclooctenes, hydrazines (e.g. hynic), aminooxy reagents, andaldehydes (e.g. 4-formyl benzoic acid).

In some embodiments, the cyclooctyne-alkyl-S—S-amine reactive groupcompounds have the structures shown in FIGS. 5-6.

In some embodiments, the cyclooctyne-alkyl-S—S-synthetic oligonucleotidecompounds have the structures represented by:

wherein RNA comprises an RNAi agent.

In some embodiments, the described cyclooctyne-alkyl-S—S-syntheticoligonucleotide compounds are used to link the oligonucleotide to anazide-containing compound. In some embodiments, the azide-containingcompound is selected from the group comprising: targeting ligand, lipid,cholesterol, or delivery agent, such as an endosomolytic polymer,polyamine, modified polyamine, or pharmacological modifier.

In some embodiments, the described cyclooctyne-alkyl-S—S-oligonucleotideis conjugated to a polymer, wherein said polymer contains an azidegroup. In some embodiments, the polymer is a polymer that facilitates invivo delivery of the oligonucleotide. In some embodiments, theoligonucleotide comprises an RNAi agent.

Disclosed herein are methods of covalently linking a syntheticoligonucleotide, such as an RNAi agent, to a pharmaceutical modifier ordelivery polymer wherein the synthetic oligonucleotide is conjugated toa described cyclooctyne-alkyl-disulfide-amine reactive group compound toform a cyclooctyne-alkyl-S—S-synthetic oligonucleotide. In someembodiments, the cyclooctyne-alkyl-S—S-synthetic oligonucleotide isfurther reacted with a pharmaceutical modifier or delivery polymer,wherein said pharmaceutical modifier or delivery polymer comprises atleast one azide group.

The described compounds and methods can be used to form RNAiagent-containing compositions that are used as therapeutic agents, asdiagnostic agents, or for use in molecular biology research.

As used herein, a cyclooctyne is a copper-free alkyne conjugationreagent. Cyclooctynes include, but are not limited to:diarylcyclooctyne, DBCO (also Azadibenzocyclooctyne (DIBAC or ADIBO)), acyclooctyne moiety fused to aryl groups (benzoannulated systems),

wherein X═C or N,

As used herein, the term “linker” means an organic moiety that connectstwo parts of a compound. Linkers typically comprise a direct bond or anatom such as oxygen or sulfur, a unit such as NR^(L) (where R^(L) ishydrogen, acyl, aliphatic or substituted aliphatic), C(O), C(O)NH, SO,SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl,arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R^(L)) (where R^(L) is hydrogen, acyl,aliphatic or substituted aliphatic), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; —(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—,—(CH₂)_(n)S—, —(CH₂)_(n)—C(O)—,—C(O)—(CH₂)_(n)—C(O)—NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(x)—,—C(O)—(CH₂)_(n)—C(O)—NH—(CH₂)_(m)——C(O)—(CH₂)_(n)—C(O)—(CH₂)_(m)—,—C(O)—(CH₂)_(n)—NH—C(O)—(CH₂)_(m)—,—C(O)—(CH₂)_(n)—O—(CH₂—CH₂—O)_(m)—(CH₂)_(x)—, —(O—CH₂—CH₂)_(n)—,—O—(CH₂—CH₂—O)_(n)—, —O—(CH₂—CH₂—O)_(n)—CH₂—, —CH₂—(O—CH₂—CH₂)_(n)—,—CH₂—(O—CH₂—CH₂)_(n)—O—, —CH₂—(O—CH₂—CH₂)_(n)—O—CH₂—,—CH₂—CH₂—(O—CH₂—CH₂)_(n)—, —(CH₂—CH₂—O)_(n)—, —(CH₂—CH₂—O)_(n)—CH₂—,

Reactive groups are those commonly available in the art and include, butare not limited to, activated ester, NHS, TFP, PFP, tetrazine,norbornenes, trans-cyclooctenes, hydrazines (e.g. hynic), aminooxyreagents, and aldehydes (e.g. 4-formyl benzoic acid).

Targeting ligands (also referred to as targeting groups) are used fortargeting or delivery of a compound to target cells or tissues, orspecific cell types. Targeting ligands enhance the association ofmolecules to a target cell. Thus, targeting ligands can enhance thepharmacokinetic or biodistribution properties of a conjugate to whichthey are attached to improve cellular distribution and cellular uptakeof the conjugate. Binding of a targeting group to a cell or cellreceptor may initiate endocytosis. Targeting groups may be monovalent,divalent, trivalent, tetravalent, or have higher valency. Targetinggroups can be, but are not limited to, compounds with affinity to cellsurface molecules, cell receptor ligands, antibodies, monoclonalantibodies, antibody fragments, and antibody mimics with affinity tocell surface molecules, hydrophobic groups, cholesterol, cholesterylgroups, or steroids. In some embodiments, a targeting group comprises acell receptor ligand. A variety of targeting groups have been used totarget drugs and genes to cells and to specific cellular receptors. Cellreceptor ligands may be, but are not limited to: carbohydrates, glycans,saccharides (including, but not limited to: galactose, galactosederivatives (such as N-acetyl-galactosamine), mannose, and mannosederivatives), haptens, vitamins, folate, biotin, aptamers, and peptides(including, but not limited to: RGD-containing peptides, RGD mimics,insulin, EGF, and transferrin).

As used herein, a steric stabilizer is a non-ionic hydrophilic polymer(either natural, synthetic, or non-natural) that prevents or inhibitsintramolecular or intermolecular interactions of a molecule to which itis attached relative to the molecule containing no steric stabilizer. Asteric stabilizer hinders a molecule to which it is attached fromengaging in electrostatic interactions. Electrostatic interaction is thenon-covalent association of two or more substances due to attractiveforces between positive and negative charges. Steric stabilizers caninhibit interaction with blood components and therefore opsonization,phagocytosis, and uptake by the reticuloendothelial system. Stericstabilizers can thus increase circulation time of molecules to whichthey are attached. Steric stabilizers can also inhibit aggregation of amolecule. A preferred steric stabilizer is a polyethylene glycol (PEG)or PEG derivative. PEG molecules suitable for the invention have about1-120 ethylene glycol monomers.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of at least one kind of RNAi agentand one or more a pharmaceutically acceptable excipients.Pharmaceutically acceptable excipients (excipients) are substances otherthan the Active Pharmaceutical ingredient (API, therapeutic product,e.g., RNAi agent) that have been appropriately evaluated for safety andare intentionally included in the drug delivery system. Excipients donot exert or are not intended to exert a therapeutic effect at theintended dosage. Excipients may act to a) aid in processing of the drugdelivery system during manufacture, b) protect, support or enhancestability, bioavailability or patient acceptability of the API, c)assist in product identification, and/or d) enhance any other attributeof the overall safety, effectiveness, of delivery of the API duringstorage or use.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, binders,buffering agents, carriers, coating agents, colors, delivery enhancers,dextran, dextrose, diluents, disintegrants, emulsifiers, extenders,fillers, flavors, glidants, humectants, lubricants, oils, polymers,preservatives, saline, salts, solvents, sugars, suspending agents,sustained release matrices, sweeteners, thickening agents, tonicityagents, vehicles, water-repelling agents, and wetting agents. Apharmaceutically acceptable excipient may or may not be an inertsubstance.

The pharmaceutical compositions can contain other additional componentscommonly found in pharmaceutical compositions. Thepharmaceutically-active materials may include, but are not limited to:anti-pruritics, astringents, local anesthetics, or anti-inflammatoryagents (e.g., antihistamine, diphenhydramine, etc.). It is alsoenvisaged that cells, tissues or isolated organs that express orcomprise the herein defined RNAi agents may be used as “pharmaceuticalcompositions”. As used herein, “pharmacologically effective amount,”“therapeutically effective amount,” or simply “effective amount” refersto that amount of an RNAi agent to produce the intended pharmacological,therapeutic or preventive result.

As used herein, a pharmacological modifier is a compound that protects,supports, or enhances stability, bioavailability, delivery,effectiveness, or patient acceptability of an API during storage or use.

As used herein a delivery polymer is a polymer that enhances in vivobioavailability or delivery of an API. Examples of delivery polymersinclude, but are not limited to: amphipathic membrane active polyamines,and reversibly modified amphipathic membrane active polyamines.

As used herein, membrane active polyamines are surface active,amphipathic polyamines that are able to induce one or more of thefollowing effects upon a biological membrane: an alteration ordisruption of the membrane that allows non-membrane permeable moleculesto enter a cell or cross the membrane, pore formation in the membrane,fission of membranes, or disruption or dissolving of the membrane. Asused herein, a membrane, or cell membrane, comprises a lipid bilayer.The alteration or disruption of the membrane can be functionally definedby the polyamine's activity in at least one the following assays: redblood cell lysis (hemolysis), liposome leakage, liposome fusion, cellfusion, cell lysis, and endosomal release. Polyamines thatpreferentially cause disruption of endosomes or lysosomes over plasmamembranes are considered endosomolytic. The effect of membrane activepolyamines on a cell membrane may be transient. Membrane activepolyamines possess affinity for the membrane and cause a denaturation ordeformation of bilayer structures. Delivery of an RNAi agent to a cellis mediated by the membrane active polyamine disrupting or destabilizingthe plasma membrane or an internal vesicle membrane (such as an endosomeor lysosome), including by forming a pore in the membrane, or disruptingendosomal or lysosomal vesicles thereby permitting release of thecontents of the vesicle into the cell cytoplasm. In some embodiments,the membrane active polyamine comprises melittin or a melittin-likepeptide (MLP).

The term polynucleotide, or polynucleic acid, is a term of art thatrefers to a polymer containing at least two nucleotides. Nucleotides arethe monomeric units of polynucleotide polymers. Polynucleotides withless than 120 monomeric units are often called oligonucleotides. Naturalnucleic acids have a deoxyribose- or ribose-phosphate backbone. Anon-natural or synthetic polynucleotide is a polynucleotide that ispolymerized in vitro or in a cell free system and contains the same orsimilar bases but may contain a backbone of a type other than thenatural ribose or deoxyribose-phosphate backbone. Syntheticoligonucleotides can be synthesized using any known technique in theart. Polynucleotide backbones known in the art include: PNAs (peptidenucleic acids), phosphorothioates, phosphorodiamidates, morpholinos, andother variants of the phosphate backbone of native nucleic acids. Basesinclude purines and pyrimidines, which further include the naturalcompounds adenine, thymine, guanine, cytosine, uracil, inosine, andnatural analogs. Synthetic derivatives of purines and pyrimidinesinclude, but are not limited to, modifications which place new reactivegroups on the nucleotide such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides. The term base encompasses any ofthe known base analogs of DNA and RNA. The term polynucleotide includesdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinationsof DNA, RNA and other natural and synthetic nucleotides.

The synthetic oligonucleotides of the invention can be chemicallymodified. The use of chemically modified polynucleotides can improvevarious properties of the polynucleotide including, but not limited to:resistance to nuclease degradation in vivo, cellular uptake, activity,and sequence-specific hybridization. Non-limiting examples of suchchemical modifications include: phosphorothioate internucleotidelinkages, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, 2′-deoxyribonucleotides, “universal base” nucleotides,5-C-methyl nucleotides, 2′,3′-seco nucleotide mimics (unlockednucleobase analogues, represented herein as N_(UNA) or NUNA), andinverted deoxyabasic residue incorporation. These chemicalmodifications, when used in various polynucleotide constructs, are shownto preserve polynucleotide activity in cells while at the same time,dramatically increasing the serum stability of these compounds.

In some embodiments, a synthetic oligonucleotide of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified, wherein each strand is about 19 to about 29 (e.g.,about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,or 29) nucleotides. In someembodiments, a synthetic oligonucleotide of the invention comprises oneor more modified nucleotides. A synthetic oligonucleotide of theinvention can comprise modified nucleotides from about 5 to about 100%of the nucleotide positions (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% ofthe nucleotide positions).

A synthetic oligonucleotide may comprise a 5′ or 3′ end modification. 3′and 5′ end modifications include, but are not limited to:amine-containing groups, alkyl groups, alkyl amine groups, reactivegroups, TEG groups, and PEG groups.

An “RNAi agent” (also called an “RNAi trigger”, or a double stranded RNAinterference polynucleotide) inhibits gene expression through thebiological process of RNA interference (RNAi). RNAi agents comprisedouble stranded RNA or RNA-like structures typically containing 15-50base pairs, and preferably 18-26 base pairs, and have a nucleobasesequence at least 85% complementary to a coding sequence in an expressedtarget gene within the cell. RNAi agents include, but are not limitedto: short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), microRNAs (miRNAs), short hairpin RNAs (shRNA), meroduplexes, and dicersubstrates (U.S. Pat. Nos. 8,084,599 8,349,809 and 8,513,207). As usedherein, the terms RNAi agent and RNAi trigger are used interchangeably.

By inhibit, down-regulate, or knockdown gene expression, it is meantthat the expression of the gene, as measured by the level of mRNAtranscribed from the gene or the level of polypeptide, protein orprotein subunit translated from the mRNA, is reduced below that observedin the absence of the compounds disclosed herein. Inhibition,down-regulation, or knockdown of gene expression, with a polynucleotidedelivered by the compositions of the invention, is preferably below thatlevel observed in the presence of a control inactive polynucleotide, apolynucleotide with a scrambled sequence or with inactivatingmismatches, or in the absence of conjugation of the polynucleotide tothe compositions disclosed herein.

In some embodiments, the RNAi agent comprises at least two sequencesthat are partially, substantially, or fully complementary to each other.In some embodiments, the two RNAi agent sequences comprise a sensestrand comprising a first sequence and an antisense strand comprising asecond sequence. In some embodiments, the two RNAi agent sequencescomprise two sense strands which together comprise a first sequence andan antisense strand comprising a second sequence, wherein the sensestrands and the antisense strand together form a meroduplex. The sensestrand may be connected to the antisense strand via a linking molecule,such as a polynucleotide linker or a non-nucleotide linker.

The antisense strand comprises a nucleotide sequence which iscomplementary to a part of an mRNA encoded by a target gene, and theregion of complementarity is most preferably less than 30 nucleotides inlength. The RNAi agent sense strands comprise sequences which have anidentity of at least 85% to at least a portion of a target mRNA. TheRNAi agent, upon delivery to a cell expressing the target gene, inhibitsthe expression of said target gene in vitro or in vivo.

In some embodiments, the RNAi agent may be comprised of naturallyoccurring nucleotides or may be comprised of at least one modifiednucleotide or nucleotide mimic. The RNAi agent sense and antisensestrands of the invention may be synthesized and/or modified by methodswell established in the art. RNAi agent nucleosides, or nucleotidebases, may be linked by phosphate-containing (natural) ornon-phosphate-containing (non-natural) covalent internucleosidelinkages, i.e. the RNAi agent may have natural or non-naturaloligonucleotide backbones. In some embodiments, the RNAi agent containsa non-standard (non-phosphate) linkage between to nucleotide bases.

As used herein, a “modified nucleotide” is a nucleotide, nucleotidemimic, abasic site, or a surrogate replacement moiety other than aribonucleotide (2′-hydroxyl nucleotide). In one embodiment a modifiednucleotide comprises a 2′-modified nucleotide (i.e. a nucleotide with agroup other than a hydroxyl group at the 2′ position of thefive-membered sugar ring). Modified nucleotides include, but are notlimited to: 2′-modified nucleotides, 2′-O-methyl nucleotides,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, 2′-methoxyethyl(2′-O-2-methoxylethyl) nucleotides, 2′-amino nucleotides, 2′-alkylnucleotides, 3′ to 3′ linkages (inverted) nucleotides, non-natural basecomprising nucleotides, bridged nucleotides, peptide nucleic acids,2′,3′-seco nucleotide mimics (unlocked nucleobase analogues, lockednucleotides, 3′-O-Methoxy (2′ intemucleotide linked) nucleotide,2′-F-Arabino nucleotides, morpholino nucleotides, vinyl phosphonatedeoxyribonucleotides, vinyl phosphonate nucleotides, and abasicnucleotides. It is not necessary for all positions in a given RNAi agentbe uniformly modified. Conversely, more than one modification may beincorporated in a single RNAi agent or even in a single nucleotidethereof. The RNAi agent sense strands and antisense strands describedherein may be synthesized and/or modified by methods known in the art.Modification at each nucleotide is independent of modification of theother nucleotides.

In some embodiments, an RNAi agent may comprise a 5′ or 3′ endmodification. 3′ and 5′ end modifications include, but are not limitedto: amine-containing groups, alkyl groups, alkyl amine groups, reactivegroups, TEG groups, and PEG groups.

In some embodiments, the RNAi agent may comprise overhangs, i.e.typically unpaired, overhanging nucleotides which are not directlyinvolved in the double helical structure normally formed by the coresequences of the herein defined pair of sense strand and antisensestrand.

In some embodiments, the RNAi agent may contain 3′ and/or 5′ overhangsof 1-5 bases independently on each of the sense strands and antisensestrands. In some embodiments, both the sense strand and the antisensestrand contain 3′ and 5′ overhangs. In some embodiments, one or more ofthe 3′ overhang nucleotides of one strand base pairs with one or more 5′overhang nucleotides of the other strand. In some embodiments, the oneor more of the 3′ overhang nucleotides of one strand do not pair withthe one or more 5′ overhang nucleotides of the other strand. The senseand antisense strands of an. RNAi agent may or may not contain the samenumber of nucleotide bases. The antisense and sense strands may form aduplex wherein the 5′ end only has a blunt end, the 3′ end only has ablunt end, both the 5′ and 3′ ends are blunt ended, or neither the 5′end nor the 3′ end are blunt ended. In some embodiments, one or more ofthe nucleotides in the overhang contains a thiophosphate,phosphorothioate, deoxynucleotide inverted (3′ to 3′ linked) nucleotide,or is a modified ribonucleotide or deoxynucleotide.

Lists of known miRNA sequences can be found in databases maintained byresearch organizations such as Wellcome Trust Sanger Institute, PennCenter for Bioinformatics, Memorial Sloan Kettering Cancer Center, andEuropean Molecule Biology Laboratory, among others. Known effectivesiRNA sequences and cognate binding sites are also well represented inthe relevant literature. RNAi agent molecules are readily designed andproduced by technologies known in the art. In addition, there arecomputational tools that increase the chance of finding effective andspecific sequence motifs (Pei et al. 2006, Reynolds et al. 2004,Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale etal. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

EXAMPLES Example 1 Synthesis of DBCO—S—S-methyl-NHS (FIG. 1, Compound 7)

A) N-Boc-Aminoacid 3: 4-(Pyridin-2-yldisulfanyl)pentanoic acid 1 (AnnovaChem., INC., product #L10067, 800 mg, 3.29 mmol) was stirred for 14 hwith 2-(boc-amino) ethanethiol 2 (1.748 g, 9.86 mmol) and DIEA (1.72 mL,9.86 mmol) in anhydrous MeOH (15 mL) under Ar at RT. The solvent wasremoved on a rotavapor, the residue was redissolved in DCM (100 mL),washed with 5% KHSO₄ (2×20 mL), H₂O (1×25 mL), and brine (1×20 mL). Thecrude product was dried over Na₂SO₄, filtered and concentrated on arotavapor. Column purification on a SiO₂ (eluent gradienthexane:EtOAc:acetic acid=9:1:0.05-7:3:0.05) yielded 650 mg (70%) of apure product 3.

B) Aminoacid 4: The product 3 (650 mg, 2.28 mmol) was stirred for 10 minin an ice-cold solution of HCl/Dioxane (4M, 6 mL) and for 1.5 h at RT.All volatiles were removed on a rotavapor, the residue was taken in DMF(2.5 mL) and crushed in Et₂O (40 mL). The separated product wastriturated with Et₂O and dried in vacuo. Yield 416 mg.

C) Compound DBCO NHS 5 was prepared in 8 steps following literatureprocedures.

D) DBCO derivative 6: The amino acid 4 (410 mg, 2.22 mmol) was stirredfor 2 h with dibenzocyclooctyne-N-hydroxysuccinimidyl (Sigma-Aldrich)(893 mg, 2.22 mmol) and DIEA (0.965 mL, 5.54 mmol) in DCM/MeOH solution(4:1, 20 mL). All volatiles were removed on a rotavap, the residual DIEAwas removed by two successive evaporations of dioxane from the crude.The product was purified on a SiO₂ column (eluent: gradient 2%-4% MeOHin CHCl₃. Yield 706 mg (68%).

E) DBCO-(monomethyl)Alkyne-S—S—NHS 7: The product 6 (700 mg, 1.48 mmol)was dissolved in anhydrous ACN (25 mL) and cooled on an ice bath. NHS(196 mg, 1.706 mmol) and EDC (326 mg, 1.706 mmol) were added and stirredfor 30 min. The ice bath was replaced and stirring was continued for 14h at RT. All volatiles were removed in vacuo, the residue wasredissolved in CHCl₃ (125 mL), washed with 2% KHSO₄ (2×25 mL), brine(2×25 mL), dried (Na₂SO₄) filtered and concentrated in vacuo. Yield 802mg (95%). (NMR: AB)

F) DBCO-dimethyl-S—S—NHS

DBCO-disulfide-dimethyl-NHS was prepared similarly to 7 using:

as a starting material.

G) Related compounds are readily made using similar techniques,including, but not limited to:

Example 2 Synthesis of DBCO-Alkyl-SMPT-NHS (Formula VI, FIG. 2 Compound13)

A) 4-bromoethylbenzoic acid (22.7 g, 0.1 mole) and thiourea (9.3 g, 0.12mol, 1.2 equiv.) were dissolved in H₂O (200 mL) and heated at reflux for4 h and then 11 h at RT. 10% NaOH (aq) (120 mL) was added and heated atreflux for 3 h. Next, the mixture was chilled to room temperature, 6 Mhydrochloric acid (60 mL) was added with the formation of theprecipitate. The precipitate was filtered, washed with cold H₂O anddried under reduced pressure. 10.4 g (58%) of crude product 8 was usedfor next step.

B) A solution of 4-mercaptoethylbenzoic acid 8 (9.60 g, 52.8 mmol) inMeOH (240 mL) was added to DPDS (23.2 g, 105.6 mmol, 2 equiv.) and AcOH(3 mL, 52.8 mmol, 1 equiv dissolved in MeOH (240 mL) over 3 h whilestirring in an ice bath. Afterwards, the reaction was brought to RT andcontinued for 2 h. Solvent was removed under reduced pressure and theremaining oil was loaded on a silica gel column. Byproducts were washedwith EtOAc:EtOH:Et₃N (200:10:2) and the product 9 (9.8 g, 64%) waseluted with EtOAc:EtOH:AcOH (200:20:1).

C) To a solution of acid 9 (9.7 g, 33.8 mmol) in MeOH (20 mL) was added2-(Boc-amino)ethanethiol (6 g, 33.8 mmol) and DIEA (5.9 mL, 33.8 mmol).The mixture was stirred under Argon for 24 h at reflux. The progress ofthe reaction was monitored by TLC. The reaction mixture was cooled toRT, concentrated and re-dissolved in EtOAc (100 mL) and washed withsaturated NaHCO₃ (3×30 mL) and brine. The EtOAc layer was dried overNa₂SO₄, concentrated and dried in vacuo to isolate crude product andfurther purified by silica gel column to give product 2A, weight 11 g,yield 91.2%. Compound 2A (11 g, 30.8 mmol) was dissolved at 0° C. in 4MHCl in dioxane (50 mL), the cooling bath was removed and the reactionwas stirred at RT for 2 h. After 2 h TLC (CHCl₃:MeOH:AcOH(9.6:0.4:0.025)) showed no starting material present. All volatiles wereremoved on a rotavap at 35° C., and then triturated with Et₂O (2×50 mL).The product 10 (as HCl salt) was dried overnight in vacuo, weight 9.1 g,yield 100%.

D) Compound DBCO NHS 11 was prepared in 8 steps following literatureprocedures.

E) DBCO NHS 11 (12.4 g, 30.8 mmol) and amine 10 (9.1 g, 30.8 mmol) weredissolved in DCM/MeOH mixture (4:1, 200 mL). DIEA (11 mL, 61.6 mmol) wasadded and the reaction mixture was stirred for 2 h under Ar. Allvolatiles were removed on a rotavap, the residue was dried on an oilpump and product was purified on SiO₂ column (eluent: gradient 1-3% MeOHin CHCl₃) to give compound 12 as a white solid, weight 11.7 g, Yield70%.

F) DBCO SMPT 13 (final product): An acid 12 (10.9 g, 20 mmol) wasdissolved in ACN/THF mixture (4:1, 150 mL) and the solution was cooledon ice bath. NHS (2.3 g, 20 mmol) and EDC (4 g, 21 mmol) weresuccessively added to the stirring reaction mixture, the cooling bathwas removed and the reaction was continued for 16 h at RT. All volatileswere removed on a rotavap, the residue was redissolved in CHCl₃ (200 mL)and washed twice with H₂O (2×50 mL) and brine (50 mL). The organic phasewas dried on MgSO₄, filtered and concentrated by a rotavap to give finalproduct DBCO SMPT(13) as white solid, weight 12.5 g, yield 97.5%.

G) Related compounds are readily made using similar techniques,including, but not limited to:

Example 3 RNAi Agents Targeting the Factor VII Gene were Conjugated toUnbranched Linkers (AD00096), Methyl Branched Linkers (AD00919), orDimethyl Branched Linkers (AD00920)

HPLC analyses (FIG. 7, 9, 10) showed significantly fewer impurities whenusing the branched linkers. Yields also improved.

A) Synthesis of DBCO-disulfide-RNAi Agent Conjugate

Crude RNA (2.7 mg, 402 nmol) with a 5′ C-6 amino modification, wasprecipitated using sodium acetate (0.5M) in EtOH, lyophilized, anddissolved in 442 μL 0.1M NaHCO₃, pH 8-9.Dibenzocyclooqyne-S—S—N-hyclroxysuccinimidyl ester (DBCO—SS—NHS) ester(2.3 mg, 4063.6 nmol) was dissolved in 227 μL DMF and added to the RNAsolution. The reaction mixture was mixed well and allowed to proceed for2 h at room temp. The reaction was monitored using RP-HPLC and thepurity was determined upon reaction completion (purity 34.4%). Afterreaction completion, the reaction mixture was dried down and purifiedusing RP-HPLC. The RNAi agent conjugate was prepared in 18% yield (75nmol). The final purity of the RNAi agent conjugate was determined byRP-HPLC (purity: 92.4%) and the identity was confirmed by MALDI-TOF/TOF(Mass calculated: 7164.0; Mass observed: 7165.3). FIG. 7. RP-HPLCchromatogram of the reaction mixture of the RNAi agent conjugateprepared with DBCO-disulfide-NHS ester (AM00253-SS, 1141-66K_6).

B) Synthesis of DBCO-RNAi Agent Conjugate

RNAi agent conjugate was similarly prepared using a DBCO—NHS esterwithout the disulfide bond. The purity upon reaction completion wasdetermined by RP-HPLC (purity: 54.0%). The RNAi agent conjugate wasprepared in 60% yield (270 nmol). The final purity was determined byRP-HPLC (purity: 90.1%) and the identity was confirmed by MALDI-TOF/TOF(Mass calculated: 6999.3; Mass observed: 6998.7). FIG. 8. RP-HPLCchromatogram of the reaction mixture of the RNAi agent conjugateprepared with DBCO—NHS ester. (AM01707-SS, 1141-18K_1).

C) Synthesis of DBCO-disulfide-methyl-RNAi Agent Conjugate

RNAi agent conjugate was similarly prepared using a DBCO—SS-methyl-NHSester with a methyl group stabilizing the disulfide bond. The purityupon reaction completion was determined by RP-HPLC (purity: 74.7%). TheRNAi agent conjugate was prepared in 53% yield (1808 nmol). The finalpurity was determined by RP-HPLC (purity: 96.6%) and the identity wasconfirmed by MALDI-TOF/TOF (Mass calculated: 7191.3; Mass observed:7191.7). FIG. 9. RP-HPLC chromatogram of the reaction mixture of theRNAi agent conjugate prepared with DBCO-disulfide-methyl-NHS.(AM01637-SS, 1141-11K_5)

D) Synthesis of DBCO-disulfide-dimethyl-RNAi Agent Conjugate

The RNAi agent conjugate was prepared using aDBCO-disulfide-dimethyl-NHS ester with two methyl groups stabilizing thedisulfide bond. The purity upon reaction completion was determined byRP-HPLC (purity: 80.6%). The RNAi agent conjugate was prepared in 66%yield (2259 nmol). The final purity was determined by RP-HPLC (purity:96.6%) and the identity was confirmed by MALDI-TOF/TOF (Mass calculated:7205.3; Mass observed: 7204.9). FIG. 10. RP-HPLC chromatogram of thereaction mixture of the RNAi agent conjugate prepared with theDBCO-disulfide-dimethyl-NHS (AM01638-SS, 1141-11K_4).

E) Synthesis of DBCO-disulfide-methyl-SMPT-RNAi Agent Conjugate

The RNAi agent conjugate was prepared using DBCO-disulfide-methyl-SMPT.The purity upon reaction completion was determined by RP-HPLC (purity:72.7%) The RNAi agent conjugate was prepared in 35% yield (1050 nmol).The final purity was determined by RP-HPLC (purity: 96.6%) and theidentity was confirmed by MALDI-TOF/TOF (Mass calculated: 7238.3; Massobserved: 7238.3). FIG. 11. RP-HPLC chromatogram of the reaction mixtureof the RNAi agent conjugate prepared with the DBCO-disulfide-methyl-SMPT(AM02455-SS, 1183-61K_2).

TABLE 1 Summary of the reaction mixture purity and percent yield usingfive different modifiers. Reaction Mixture Percent Yield after Type ofModifier Purity Purification DBCO-disulfide-NHS 34.4 18.8 DBCO-NHS 54.060.3 DBCO-disulfide-methyl-NHS 74.7 52.8 DBCO-disulfide-dimethyl-NHS80.6 65.9 DBCO-disulfide-methyl-SMPT 72.7 35.4

In stabilizing the disulfide bond, the RNAi agent conjugate can beprepared with greater purity before purification, allowing for a greaterreaction yield.

LC showed decreased impurities using branched linkers (DBCO conjugatedto RNAi agent)

Example 4 In Vivo Effectiveness of Alkyne-disulfide-alkyl Modifiers

A) RNAi agents. of the following sequences were synthesized usingstandard phosphoramidite chemistry with C6 amino linker at the 5′ end ofthe sense strand added using commercially-available6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(from Glen Research).

TABLE 2 Factor VII RNAi agents Duplex SEQ ID strand ID Sequence 5′→3′AD00096 Antisense  1 dTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT Sense  2(DBCO-SS-C6)GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) AD00919 Antisense  3dTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT Sense  4(DBCO-SS-methyl-C6)GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) AD00920Antisense  5 dTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT Sense  6(DBCO-SS-dimethyl-C6)GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) AD00921Antisense  4 dTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT Sense  8(DBCO-C6)GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) AD01477 Antisense  9dTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT Sense 10(DBCO-SMPT-C6)GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) n (lower case) =2'-OMe substitution nucleotide Nf = 2'-Fluoro nucleotide dN = 2-deoxysubstitution; inv = 3' to 3' linkage s = phosphorothioate linkage

B) RAFT copolymer of N-Boc-ethoxyethylamine acrylate and sec-butylacrylate (EAB). Solutions of AIBN (1.00 mg/mL) and RAFT agent CPCPA(10.0 mg/mL) in butyl acetate were prepared. Monomer molar feed was 55%N-Boc-ethoxyethylamine acrylate, 45% sec-butyl acrylate (CAS#2998-08-5). Theoretical Mw was 100,000.

N-Boc-ethoxyethylamine acrylate (0.890 g, 3.43 mmol) sec-butyl acrylate(0.391 mL, 0.360 g, 2.81 mmol) CPCPA solution (0.350 mL, 0.0125 mmol),AIBN solution (0.308 mL, 0.00188 mmol), and butyl acetate (5.3 mL) wereadded to a 20 mL glass vial with stir bar. The vial was sealed with asepta cap and the solution bubbled with nitrogen using a long syringewith a second syringe as the outlet for 1 h. The syringes were removedand the vial heated to 80° C. for 16 h using an oil bath. The solutionwas allowed to cool to room temperature and transferred to a 50 mLcentrifuge tube before hexane (35 mL) was added to the solution. Thesolution was centrifuged for 2 min at 4,400 rpm. The supernatant layerwas carefully decanted and the bottom (solid or gel-like) layer wasrinsed with hexane. The bottom layer was then re-dissolved in DCM (7mL), precipitated in hexane (40 mL) and centrifuged once more. Thesupernatant was decanted and the bottom layer rinsed with hexane beforethe polymer was dried under reduced pressure for several hours. Yield ofcrude EAB copolymer was 0.856 g. Samples of the crude polymer were takenfor multi-angle light scattering (MALS). The dried, crude copolymer wasdissolved in DCM (100 mg/mL). Hexane was added until just after thecloud point was reached. The resulting milky solution was centrifuged.The bottom layer was extracted and fully precipitated into hexane. Thefraction was centrifuged, after which the copolymer was isolated anddried under vacuum. Yield of isolated fraction of EAB copolymer was0.478 g. Samples of the fractionated copolymer were taken for ¹H-NMR andMALS. Composition determined by ¹H-NMR was 61% N-Boc-ethoxyethylamineand acrylate, 39% sec-butyl acrylate.

MALS Analysis. Approximately 10 mg of the copolymer was dissolved in 0.5mL 75% dichloromethane, 20% tetrahydrofuran, 5% acetonitrile. Themolecular weight and polydispersity (PDI) were measured using a WyattHeleos II multiangle light scattering detector attached to a ShimadzuProminence HPLC using a Jordi 5μ 7.8×300 Mixed Bed LS DVB column. CrudePolymer: MW: 59,000 (PDI 1.3), Fractionated Polymer: MW 70,000 (PDI:1.1).

Deprotection/Dialysis. The dried samples were treated with 2M HCl inacetic acid (˜7 ml) for 1 h to remove the BOC protecting groups. Thenthe reaction was diluted with 20 mL of water and allowed to stir for10-15 min. The fractions were then dialyzed with 3500 MW dialysis tubingin high salt, high salt, and water for 15 h, 8 h, and 15 h respectively.The fractions were then transferred to 50 mL centrifuge tubes andlyophilized for 3 days or until dry. The dry samples were brought up at20 mg/mL in water for further study.

RNAi agent-polymer formulations. Polyacrylate EAB in 5 mM pH 8.0 HEPESbuffer was modified 1.5 wt % with a N-hydroxysuccinimidyl activated PEG₄azide (Azido-dPEG₄-NHS ester from Quanta Biodesign) to provide azidegroups for subsequent attachment of RNAi agent. The azide-modifiedpolymer was then diluted to 5 mg/mL in 60 mg/mL HEPES base. To thissolution was added 15 mg/mL (3 wt equivalents) PEG masking reagentPEG-Ala-Cit-PABC to modify 40-50% of available amine groups. After 1 h,DBCO-modified rodent Factor VII RNAi agent (0.125 wt eq relative topolymer) was added to polymer solution. After incubation overnight,conjugates were further modified by addition of molar excess relative toavailable amine groups of an N-acetylgalactosamine derivativeNAG-Ala-Cit-PABC (presented in Table 2), and incubated for 30 minutes.

Mice and injection procedures. Female ICR mice, 6 to 8 weeks old, wereobtained from Harlan Sprague-Dawley, (Indianapolis, Ind.). All the micewere handled in accordance with animal used protocols approved by theAnimal Care and Use Committee at Arrowhead Madison Inc. Rodents weremaintained on a 12 h light/dark cycle with free access to water and food(Harlan Teklad Rodent Diet, Harlan, Madison, Wis.). Deliveryformulations were injected as a bolus into the tail vein in a totalvolume of 0.2 mL (mice) or HEPES-buffered (5 mM, pH 7.5) isotonicglucose under standard conditions. Mice were injected with 2 mg/kgpolymer conjugated to 0.25 mg/kg RNAi agent. Serum samples collected 5days post-injection.

Serum FVII activity measurements. Serum samples were prepared bycollecting blood by submandibular bleeding into microcentrifuge tubescontaining 0.109 M sodium citrate anticoagulant (1 volume) followingstandard procedures. FVII activity in serum was measured with achromogenic method using a test kit (BIOPHEN VII, Aniara, Mason, Ohio)following manufacturer's recommendations. Absorbance of colorimetricdevelopment was measured using a Tecan Safire-2 microplate reader at 405nm.

TABLE 3 Normalized Factor VII levels after injection of polymer-RNAiagent disulfide conjugates. DBCO disulfide Relative F VII activityEAB-RNA  91 ± 15 EAB-SS-RNA 16 ± 7 EAB-SS-methyl-RNA 12 ± 7EAB-SS-dimethyl-RNA  36 ± 10 EAB-S-S-methyl-SMPT-RNA 26 ± 7

1. An RNAi agent-containing composition for inhibiting the expression ofa target gene in a cell in vitro or in vivo, the RNAi agent-containingcomposition comprising an RNAi agent covalently linked to ancyclooctyne-alkyl disulfide compound having the structure representedby:

wherein R¹ is an alkyl group and R² is hydrogen, or R¹ is hydrogen andR² is an alkyl group, L¹ and L² are linkers, X comprises a cyclooctyne,and Y comprises a reactive group.
 2. The RNAi agent-containingcomposition of claim 1, wherein the alkyl group is a methyl group. 3.The RNAi agent-containing composition of claim 1, wherein X isdiarylcyclooctyne, DBCO, Azadibenzocyclooctyne (DIBAC or ADIBO), acyclooctyne moiety fused to aryl groups,

wherein X═C or N,


4. The RNAi agent-containing composition of claim 1, further includingone or more pharmaceutically acceptable excipients.
 5. The RNAiagent-containing composition of claim 1, wherein the RNAi agent is ansiRNA.
 6. An RNAi agent-containing composition for inhibiting theexpression of a target gene in a cell in vitro or in vivo, the RNAiagent-containing composition comprising an RNAi agent covalently linkedto an cyclooctyne-alkyl disulfide compound having the structurerepresented by:

wherein R¹ and R^(1′) are each alkyl groups and R² and R^(2′) are eachhydrogen, or R¹ and R^(1′) are each hydrogen and R² and R^(2′) are eachalkyl groups, L¹ and L² are linkers, X comprises a cyclooctyne, and Ycomprises a reactive group.
 7. The compound of claim 6, wherein thealkyl groups are methyl groups.
 8. The RNAi agent-containing compositionof claim 6, wherein X is diarylcyclooctyne, DBCO, Azadibenzocyclooctyne(DIBAC or ADIBO), a cyclooctyne moiety fused to aryl groups,

wherein X═C or N,


9. The RNAi agent-containing composition of claim 6, further includingone or more pharmaceutically acceptable excipients.
 10. The RNAiagent-containing composition of claim 6, wherein the RNAi agent is ansiRNA.
 11. A method of inhibiting the expression of a target gene in acell, the method comprising delivering to the cell an effective amountof the composition of claim
 1. 12. A method of inhibiting the expressionof a target gene in a cell, the method comprising delivering to the cellan effective amount of the composition of claim 6.